Fibrinogen is one of the major coagulation factors in the vertebrates.
Measure of its content in an individual can provide different information and/or be the function of many variables. For example, fibrinogen level is increased in case of acute phase reaction or in post-menopausal women, and also increases with ageing, pregnancy or oral contraception. On the contrary, fibrinogen level is decreased in case of liver diseases, inherited deficiencies such as hypofibrinogenaemia and dysfibrinogenaemia, disseminated intravascular coagulopathy or during massive bleeding.
More particularly, due to its essential role in blood clotting, the fibrinogen content of an individual suffering or likely to suffer of a severe haemorrhage needs to be evaluated as fast as possible, notably as it is an early predictor of outcome. The result obtained, as well as the measurement of the haematocrit of the individual, are critical information for determining whether this individual has developed a coagulopathy and needs to receive immediately coagulation factors supplement and/or massive transfusion. Thus, fibrinogen concentration appears as a trigger for initiating replacement as well as for a treatment goal.
Indeed, a low concentration of fibrinogen in the blood can be associated with an increase of the risk of haemorrhage and/or an increase of the on-going bleeding. Moreover, it has been demonstrated that in case of haemorrhage, fibrinogen is the first coagulation factor to reach critical level. This decrease is mostly due to its conversion into fibrin by thrombin during blood clot formation.
Several methods well known in the art are already available to estimate fibrinogen content in plasma. Mention can be made of the Prothrombin Time-derived fibrinogen assay, in which Prothrombin Time is determined by optical density change for a range of plasma dilutions with known fibrinogen levels, and the result is obtained on a reference curve. Well known immunological assays are also used, such as ELISA assay, radial immunodiffusion or electrophoresis. Different gravimetric assays, comprising Jacobsson's method, are also commonly used and are based on the formation of a clot by addition of thrombin or reptilase in excess before washing, drying then weighting the obtained clot. An alternative consists in dissolving the clot after its formation in an urea-alkaline solution then performing spectrophotometry in order to determine the concentration of fibrin through its extinction coefficient, considering that all the fibrinogen has been converted in fibrin and that the clot is only constituted of said fibrin.
Nowadays, the most commonly used method for determining fibrinogen content in an individual is the Clauss method. This method is carried out on diluted plasma, and consists in measuring the time to clot formation following addition of an excess of thrombin (typically 100 U/ml), so that the clotting time is independent of the thrombin concentration. Time to clot is detected photometrically by means of turbidity increase or mechanically through the cease of movement resulting from the gelation using a signal provided by a magnetic rod or added particles (Oberhadt et al., Clin. Chem. (1991), 37:520-526). A reference curve allows deducing the fibrinogen content as a function of the time to clot formation.
However, these methods are usually carried out on plasma in a laboratory. Then, they all require first separating it from the whole blood of the individual to be tested by centrifugation before performing the fibrinogen content assay.
Moreover, if the gravimetric assays can be considered as being more accurate than the Clauss method, it requires at least 2 hours to provide a result, whereas the Clauss method still necessitates 20 minutes including plasma separation. It can further be noted that these methods necessitate the use of a bulky and expensive equipment. These methods are also time consuming and are not accurate in case of massive bleeding.
Hence, in the field of emergency care, there is a need for a method appropriate for the assessment within minutes of bleeding risks and in the therapeutic management of bleeding based on the measure of fibrinogen level.
Also, there is a need for a method allowing within minutes the detection of an alarming fibrinogen level and in particular less than 1.5 g/L.
Moreover, there is a fundamental need to develop a method that can provide within minutes, in particular in less than 5 minutes, more particularly within 3 minutes, a sufficiently reliable result based on the measure of fibrinogen level to determine if an individual needs to receive coagulation factors supplement and/or to be transfused.
There is further a need for a method only necessitating a transportable material, in particular in an ambulance or any emergency rescue vehicle, or that can even be transportable by rescuers and emergency care personnel, for example in a specialised suitcase or backpack.
Furthermore, as all the methods used to date by care professionals were based on plasma fibrinogen content, the reference values concerning fibrinogen level are internationally based on plasma fibrinogen content. There is thus a need for a method based on a whole blood sample instead of plasma, combined with a mean to transcribe the results obtained in plasma fibrinogen content.
Consequently, there is a need for a method allowing within minutes to determine both haematocrit and fibrinogen content in whole blood of an individual.
Haematocrit is the percentage of red blood cells in the whole blood of an individual. Measure of its level in an individual can provide different information. For example, an increase of haematocrit is found in people suffering from dehydration, oxygen deprivation or polycythemia. On the contrary, a decrease of the haematocrit occurs during anaemia. Anaemia can result from different causes, such as cancer, cirrhosis or vitamin deficiency, but is also a sign of blood loss, and as such internal bleeding.